Polyomavirus middle T (MT) is responsible for murine polyomavirus (PyV) tumorigenesis. Otherwise wild type viruses that have mutant MT fail to induce tumors. Transgenic MT causes tumors in many tissues, providing valuable models for diseases such as breast cancer. Studies of MT led to the discovery of phosphoinositide 3-kinase (PI3K) and protein tyrosine phosphorylation. Much evidence suggests that continued study of MT will yield new insights into neoplastic transformation and tumorigenesis. Our central hypothesis is that MT promotes transformation by the modulation of specific steps in cell signaling that affect not only the tumor cell itself, but also associated stromal cells, to support tumor progression. We will focus on three areas where MT studies can offer novel insight into neoplastic transformation: 1) Protein phosphatase 2A (PP2A): The role we have uncovered for the A? isoform of PP2A in activation of c- src by MT points to the importance of specific isoforms. We will examine the role of both A subunit isoforms of PP2A and loss of specific trimeric ABC complexes of PP2A in MT transformation. We have evidence for a new model whereby MT brings substrates to PP2A for dephosphorylation. The lipins, which are both enzymes of the lysophosphatidic acid (LPA) metabolism and also transcriptional coactivators, are examples of such substrates that point to novel PP2A-dependent signaling in transformation. We will test their role in transformation. Guided by a mutant defective in lipin binding, we will also use mass spectrometry of mutant and wild type (WT) MT to uncover additional substrates. 2) PI3K: MT mutant E349K shows that there is a step, so far unappreciated, between PI3K recruitment to activated tyrosine kinases and signaling to activate downstream targets such as Akt. The nature of the defect will be determined in studies of PI lipids and protein localization. Using mass spectrometry comparing E349K to WT, we will seek to identify the cellular protein(s) responsible. Identification of a new intermediate in PI3K signaling would be paradigm shifting and offer new possibilities for therapeutic intervention. 3) Cytokines: Although cytokine expression is well recognized to be an important part of tumorigenesis, little progress has been made in understanding the signals that control the patterns of cytokine expression. More importantly, the idea that cancer signaling alters responses to cytokines is largely unexplored. Our evidence shows that MT both induces cytokine expression and at the same time alters the response of tumor cells to cytokines. We will determine the spectrum of effects on cytokine expression and response that MT has both in tumor cells and on stromal cells by gene array and protein analysis. Using genetic tools we will investigate the mechanisms MT uses to do this and carry out novel tests of how the genetic background of the host modulates responses. Finally, we will ask how these effects determine macrophage recruitment in vitro and tumorigenesis in vivo studies. Our work will thus close gaps in our knowledge of cytokines and determine their contribution to MT mammary tumorigenesis. PUBLIC HEALTH RELEVANCE: Widespread in human and animal populations, polyomaviruses have provided fundamental insights into growth regulation of normal and cancer cells. Using these viruses, we seek to identify new pathways responsible for tumor growth, invasion and metastasis. We expect this will improve our understanding of cancer and offer new possibilities for its treatment.